Methods to identify combinations of ns5a targeting compound that act synergistically to inhibit hepatitis c virus replication

Abstract

The present invention is based on the surprising finding that pairs of HCV NS5A-targeting inhibitors can be identified which display similar resistance profiles yet, when combined, exhibit synergistic inhibition of wild type replicons and/or replicons carrying mutations conferring resistance to the HCV NS5A-targeting inhibitor. In addition, combinations of these molecules result in a higher genetic barrier to resistance, demonstrating their potential utility as novel combination therapies for treatment of HCV.

1 . A method for identifying NS5A-targeting compounds that in combinations demonstrate a synergistic inhibitory interaction toward wild-type and/or variants that reduce the potency of the NS5A-targeting inhibitors alone comprising the steps of:
 (a) screening a compound library for NS5A targeting compounds and   (b) comparing the amount of HCV inhibition of a NS5A-targeting inhibitor in the presence and absence of a fixed concentration of a NS5A-targeting compound and   wherein the second NS5A-targeting compound may or may not demonstrate HCV NS5A inhibitory activity when assayed alone and wherein the combination of the NS5A-targeting compounds demonstrate a synergistic inhibition of HCV replicon or virus replication.   
     
     
         2 . The method of  claim 1  wherein the compound library is screened in cells containing a HCV sequence derived from any genotype. 
     
     
         3 . The method of  claim 1  wherein the HCV inhibitory activity is determined by using a cell-based method or a biochemical surrogate. 
     
     
         4 . The method of  claim 2  wherein the HCV sequence is selected from the group consisting of wild-type or sequences carrying NS5A resistant variants. 
     
     
         5 . The method of  claim 2  wherein said HCV sequence is genotype 1b carrying a Y93H single amino acid substitution in NS5A. 
     
     
         6 . The method of  claim 2  wherein said HCV sequence is genotype 1b carrying a L31V single amino acid substitution in NS5A. 
     
     
         7 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying a M28T single amino acid substitution in NS5A. 
     
     
         8 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying a Q30R single amino acid substitution in NS5A. 
     
     
         9 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying a L31V single amino acid substitution in NS5A. 
     
     
         10 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying a Y93H single amino acid substitution in NS5A. 
     
     
         11 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying a Q30H single amino acid substitution in NS5A. 
     
     
         12 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying a Q30E single amino acid substitution in NS5A. 
     
     
         13 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying a Y93N single amino acid substitution in NS5A. 
     
     
         14 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both L31V and Y93H in NS5A. 
     
     
         15 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both M28T and Q30H in NS5A. 
     
     
         16 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both Q30R and H58D in NS5A. 
     
     
         17 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both Q30H and Y93H in NS5A. 
     
     
         18 . The method of  claim 2  wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both Q30R and E62D in NS5A. 
     
     
         19 . The method of  claim 2  wherein said HCV sequence is genotype 1b carrying amino acid substitutions at both L31V and Y93H in NS5A. 
     
     
         20 . The method of  claim 3 , wherein said cell-based method is a transient replication assay. 
     
     
         21 . The method of  claim 3 , wherein said cell-based method is a FRET assay. 
     
     
         22 . The method of  claim 3 , wherein said cell-based method is a luciferase assay. 
     
     
         23 . The method of  claim 3 , wherein said cell-based method is a colony formation assay. 
     
     
         24 . The method of  claim 3 , wherein said cell-based method is a Western blot assay. 
     
     
         25 . The method of  claim 3 , wherein said cell-based method is a Taqman assay. 
     
     
         26 . The method of  claim 3 , wherein said cell-based method is an ELISA assay. 
     
     
         27 . The method of  claim 3 , wherein said biochemical surrogate is a NS5A hyperphosphorylation assay. 
     
     
         28 . The method of  claim 1 , wherein the HCV inhibitory activity is increased by at least 3 fold.